Method for production of beta-cryptoxanthin and alpha-cryptoxanthin from commerically available lutein

ABSTRACT

The present invention relates to a method of reacting (3R,3′R,6′R)-lutein with a catalytic amount of an acid to obtain a mixture of anhydroluteins, rich in anhydrolutein III, with substantially no Z-isomers being formed. The mixture is converted to (3R)-β-cryptoxanthin (major product) and (3R,6′R)-α-cryptoxanthin (minor product) by reacting the anhydroluteins with borane-amine complexes (e.g. Me 3 N.BH 3 ) or other hydride donors and an acid in a chlorinated solvent, preferably dichloromethane, at ambient temperature to produce (3R)-β-cryptoxanthin and (3R,6′R)-α-cryptoxanthin.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is in the field of organic chemistry. The inventionrelates to an efficient process for transforming commercially available(3R,3′R,6′R)-lutein containing 5-7% (3R,3′R)-zeaxanthin toanhydroluteins (dehydration products of lutein) in an alcohol atelevated temperatures and subsequent conversion of the latter to(3R)-β-cryptoxanthin as the major product and (3R,6′R)-α-cryptoxanthinas the minor product.

2. Related Art

A process for converting commercially available (3R,3′R,6′R)-lutein to amixture of (3R,6′R)-α-cryptoxanthin, (3R)-β-cryptoxanthin, and(3R,6′R)-anhydrolutein I ((3R,6′R)-3′,4′-didehydro-β,γ-caroten-3-ol),(3R,6′R)-2′,3-anhydrolutein II((3R,6′R)-2′,3′-didehydro-β,ε-caroten-3-ol), and(3R)-3′,4′-anhydrolutein III ((3R)-3′,4′-didehydro-β,β-caroten-3-ol) inone synthetic step by allylic deoxygenation with a strong acid and ahydride ion donor was described by Khachik in U.S. Patent ApplicationNo. 60/220,995. The chemical structures of these carotenoids are shownin Scheme 1.

Khachik also described a two-step alternative process. The first stepconverted (3R,3′R,6′R)-lutein to a mixture of anhydroluteins I, II, IIIat room temperature with an acid. In the second step, the isolatedanhydroluteins were converted to (3R,6′R)-α-cryptoxanthin and(3R)-β-cryptoxanthin with a strong acid and a hydride ion donor.

As described by Khachik, the acid-catalyzed dehydration of(3R,3′R,6′R)-lutein in a homogenous phase in a variety of solvents suchas ethers (tetrahydrofuiran, tert-butyl methyl ether), chlorinatedsolvents (dichloromethane, chloroform, 1,2-dichloroethane), acetone, andtoluene at ambient temperature leads to the formation of considerableamount of Z(cis)-isomers of anhydroluteins. In addition, under theconditions disclosed in '995 application, anhydrolutein I is the majorproduct and anhydroluteins II and III are the minor products. Becauseanhydrolutein III is the precursor to (3R)-β-cryptoxanthin in the ionichydrogenation step, a higher concentration of this carotenoid relativeto anhydroluteins I and II is preferred. Therefore a modified procedurefor the dehydration of (3R,3′R,6′R)-lutein is needed that can produceanhydrolutein III as the major product and at the same timesignificantly reduce E/Z(trans/cis)-isomerization of anhydroluteins.Such a procedure must also demonstrate that a mixture of anhydroluteinswith high concentration of anhydrolutein III can be transformed into amixture of all-E-cryptoxanthins with a high concentration ofall-E-(3R)-β-cryptoxanthin. Since (3R)-β-cryptoxanthin is a precursor ofvitamin A, a higher concentration of this carotenoid in the finalproduct is desirable.

SUMMARY OF THE INVENTION

To substantially increase the yield of (3R)-β-cryptoxanthin relative to(3R,6′R)-α-cryptoxanthin in the final product and at the same timeprevent any significant E/Z(trans/cis)-isomerization of the resultingcarotenoids, (3R,3′R,6′R)-lutein is first dehydrated in the presence ofcatalytic amounts of an acid (e.g. sulfuric acid, hydrochloric acid) inan alcohol (e.g. ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol,isobutyl alcohol, tert-butyl alcohol, 1-pentanol (n-amyl alcohol),2-pentanol, n-hexyl alcohol, n-octyl alcohol, ethylene glycol, propyleneglycol) at 78-87° C. to give a mixture of anhydrolutein I as majorproduct and anhydroluteins II and III as minor products. As heating iscontinued anhydrolutein I gradually undergoes isomerization at 78-87° C.to anhydrolutein III. However, during this process considerable amountof Z(cis)-isomers of anhydroluteins are formed. To revert theZ(cis)-isomers of anhydroluteins to all-E(trans)-isomers, water is addedand heating at 78-87° C. is continued until the product comprisessubstantially all-E-isomers of anhydroluteins. Anhydrolutein III servesas a precursor to (3R)-β-cryptoxanthin in the ionic hydrogenation step.

In an alternative process, (3R,3′R,6′R)-lutein is allowed to react withan alcohol, used as solvent, in the presence of catalytic amount of anacid between 45-50° C. to give the corresponding 3′-alkyl ethers oflutein (Scheme 2). Because the temperature is maintained below 50° C.,lutein 3′-alkyl ethers do not undergo acid-catalyzed elimination to giveanhydroluteins and at the same time E(trans)/Z(cis)-isomerization isalso suppressed. Water and additional acid is then added to the mixtureand the temperature is raised to 78-87° C. to convert the lutein3′-alkyl ethers to a mixture of anhydroluteins I, II, and III,quantitatively. At the beginning of this transformation, once againanhydrolutein I is the major product and anhydrolutein II and III arethe minor products. As heating continues at 78-87° C., anhydroluteins Iand II are partially isomerized to anhydrolutein III within 7-20 hdepending on the nature of the alcohol (Scheme 2).

Anhydroluteins prepared by both methods may be simply removed byfiltration and then subjected to ionic hydrogenation with or withoutpurification. In this step, the dehydration products of(3R,3′R,6′R)-lutein, comprising anhydrolutein III as the major product,are treated with a strong acid and a hydride ion donor to produce amixture of all-E(trans)- and Z-isomers of (3R)-β-cryptoxanthin (majorproduct) and (3R,6′R)-α-cryptoxanthin (minor product) in excellentyields. Using the same strategy described in preparation ofall-E(trans)-anhydroluteins, the mixture of (E/Z)-cryptoxanthins isheated in an alcohol at 78-87° C. to revert the Z(cis)-isomers of(3R)-β-cryptoxanthin and (3R,6′R)-α-cryptoxanthin to their all-E(trans)compounds. The products formed according to this procedure do notcontain any significant E/Z(trans/cis)-isomers and more than 90% ofanhydroluteins are converted to all-E-(3R)-β-cryptoxanthin andall-E-α-cryptoxanthin.

In a preferred embodiment, the present invention relates to a processfor converting (a) (3R,3′R,6′R)-lutein (85% total carotenoids) or (b)purified lutein (97% total carotenoids) containing 5-7%(3R,3′R)-zeaxanthin or mixtures of (a) and (b) to a mixture ofanhydroluteins I, II, and III at elevated temperature, comprisingreacting (3R,3′R,6′R)-Autein in an alcohol or mixture of alcohols with acatalytic amount of an aqueous mineral acid or a strong organic acid atan elevated temperature to give a mixture of anhydroluteins comprisinganhydrolutein I as the major product, anhydroluteins II and III as theminor products, and the recovered (3R,3′R)-zeaxanthin. According to thisprocess, it is possible to obtain a mixture of anhydroluteins comprisingat least 80% anhydrolutein III and substantially no Z-anhydrolutein III.

In an alternative embodiment, the present invention relates to a processfor converting (a) (3R,3′R,6′R)-lutein (85% total carotenoids) or (b)purified lutein (97% total carotenoids) containing 5-7%(3R,3′R)-zeaxanthin or mixtures of (a) and (b) to lutein 3′-alkyl ether,comprising reacting (3R,3′R,6′R)-lutein with an alcohol in the presenceof a catalytic amount of an aqueous mineral acid or a strong organicacid at an elevated temperature to give all-E(trans)-lutein 3′-alkylether and the recovered (3R,3′R)-zeaxanthin.

In an alternative embodiment, the present invention relates to A processfor converting a mixture of all-E-anhydroluteins rich inall-E-anhydrolutein III, to a mixture of all-E-(3R)-β-cryptoxanthin andall-E-(3R,6′R)-α-cryptoxanthin, comprising reacting anhydroluteinscontaining 3-8% (3R,3′R)-zeaxanthin in a chlorinated solvent with about1.3 equivalent of a hydride donor and about 3.5-4 equivalent of a strongorganic acid at ambient temperature for about 1-5 hours to give amixture of E/Z-(3R)-β-cryptoxanthin, E/Z-(3R,6′R)-α-cryptoxanthin,unreacted E/Z-anhydroluteins, and recovered E/Z-(3R,3′R)-zeaxanthin.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention, commercially available(3R,3′R,6′R)-lutein (containing 5-7% (3R,3′R)-zeaxanthin) reacts with analcohol, employed as solvent, in the presence of catalytic amount of anacid at 45-50° C. to give the corresponding lutein 3′-alkyl ethers(Scheme 2). At the beginning of the acid-catalyzed dehydration reaction,(3R,3′R,6′R)-lutein exists as a suspension in alcohol. However, once thecorresponding lutein 3′-alkyl ethers are formed, a uniform solution isobtained. If the temperature of the solution is increased above 50° C.,acid-catalyzed elimination of alcohol from lutein 3′-alkyl ethersresults in a mixture of anhydroluteins in which anhydrolutein I is themajor product and anhydroluteins II and III are the minor products.Further heating of this solution between 78-87° C. results inacid-catalyzed isomerization of anhydroluteins I and II to anhydroluteinIII. In this homogenous solution, the isomerization is also accompaniedby the formation of substantial amounts of Z(cis)-anhydroluteins. TheseZ(cis)-anhydroluteins are converted to their all-E-isomers in aheterogeneous system in the presence of water when heated between 78-87°C.

In an alternative embodiment, the lutein 3′-alkyl ethers are formedbelow 50° C. and water is added to lower the solubility of lutein3′-alkyl ethers. Additional amounts of acid is added and the reactiontemperature raised to about 78-87° C. to form the dehydration productsof (3R,3′R,6′R)-lutein with the loss of the corresponding alcohol. Underthese conditions, anhydrolutein I is formed as the major product andanhydroluteins II and III are the minor products. After continuousheating at an elevated temperature in the range of 78-87° C. for up to7-20 h, anhydrolutein I and II gradually undergo isomerization to formanhydrolutein III. Substantially no E/Z(trans/cis)-isomerization ofthese carotenoids at elevated temperatures takes place. If water isadded at the beginning of the reaction, the dehydration of(3R,3′R,6′R)-lutein does not proceed to completion and results in anepimeric mixture of (3R,3′R,6′R)-lutein and (3R,3′S,6′R)-lutein(3′-epilutein).

In an alternative embodiment of the invention, a combination of theabove methods are employed to reduce the reaction time. In thisapproach, a considerable amount of anhydrolutein III is prepared at78-87° C. before addition of water (method 1) and then water andadditional amounts of acid is added and heating is continued to completethe conversion of anhydroluteins I and II to anhydrolutein III (Method2).

Depending on the nature and the boiling point of the alcohol, length ofheating, and the concentration of acid, the relative ratio ofanhydrolutein III to anhydrolutein I and II can be readily manipulatedby the methods described earlier. During these processes, anhydroluteinsI and II are not completely isomerized to anhydrolutein III. Forexample, when 1-propanol is used as the alcohol and the reaction mixtureis heated at 87-87° C. for 20 h by Method 2, the relative distributionof anhydroluteins is: anhydrolutein III (84%), anhydrolutein I (10%),and anhydrolutein II (6%). The present invention will furtherdemonstrate that a mixture of anhydrolutein with a high concentration ofanhydrolutein III can be converted to a mixture of all-E-cryptoxanthinswith a high concentration of (all-E, 3R)-β-cryptoxanthin.

In the second step, the dehydration products of (3R,3′R,6′R)-luteinundergo ionic hydrogenation with a strong acid and a hydride ion donorat ambient temperature to yield (3R)-β-cryptoxanthin and(3R,6′R)-α-cryptoxanthin as major and minor products, respectively. Thisstep can be carried out with the crude product after removal of waterand alcohols by filtration or alternatively anhydroluteins can be firstisolated and purified prior to ionic hydrogenation.

The commercially available (3R,3′R,6′R)-lutein employed in thesereactions may be isolated from extracts of marigold flowers and maycontain approximately 5-7% (3R,3′R)-zeaxantlhin. Because(3R,3′R)-zeaxanthin does not react with acids or hydride iondonors/acid, this carotenoid can be recovered in the final product.

Reagents and Starting Materials

Two types of (3R,3′R,6′R)-lutein may be employed as starting materialsin this invention, these are: 1) commercially available(3R,3′R,6′R)-lutein with approximately 85% total carotenoid purity and2) crystalline lutein with greater than 97% total carotenoid purityaccording to the process described in WO 99/20587. Both startingmaterials are prepared from crude saponified extracts of Marigoldflowers and contain approximately 5-7% (3R,3′R)-zeaxanthin. Mixtures ofthese two starting materials may also be employed.

The crude saponified extract of Marigold flower containing(3R,3′R,6′R)-lutein and several minor carotenoids may be preparedaccording to the process described in WO 99/20587. (3R,3′R,6′R)-Lutein(97% total carotenoid purity) containing approximately 5-7% zeaxanthinmay also be purified from this extract according to this procedure.Commercially available (3R,3′R,6′R)-lutein (85% total carotenoid) may beobtained from Kemin Industries (Des Moines, Iowa). All reagents used inthis invention are commercially available (Aldrich Chemical Co.,Milwaukee, Wis.) and are used without further purification. Thecarotenoid composition of the 85% and 97% lutein is shown in Table 1.TABLE 1 Carotenoid composition of 85% and 97% (3R,3′R,6′R)-luteinisolated from marigold flowers. Composition 85% total 97% total MarigoldCarotenoids carotenoid purity carotenoid purity(all-E,3R,3′R,6′R)-lutein^(a) 91.0 95.0 (all-E,3R,3′R)-zeaxanthin 6.545.0 Anhydroluteins (lutein dehydration 0.43 0.0 products) β-carotene0.35 0.0 α-cryptoxanthin 0.41 0.0 β-cryptoxanthin 0.38 0.03-hydroxy-β,ε-caroten-3′-one 0.89 0.0 Total 100.0 100.0^(a)The 85% and 97% lutein did not contain any significant amount of Z(cis)-luteins. Acid-Catalyzed Dehydration of (3R,3′R,6′R)-Lutein toAnhydroluteins at Elevated Temperatures by Method 1

In a typical experiment, a suspension of 85% commercially available(3R,3′R,6′R)-lutein (1.0 g of 85% pure, 0.85 g, 1.49 mmol) in 30 ml ofan alcohol is treated with 0.2-0.8 ml of 50% sulfuric acid/water (v/v).The mixture is heated to an elevated temperature. The mixture is heatedat the elevated temperature of about 78-87° C. for 0.5-4 h. Depending onthe nature of the alcohol and the length of heating, (3R,3′R,6′R)-luteinis converted to a mixture of E/Z(trans/cis)-anhydroluteins in whichE/Z(trans/cis)-anhydrolutein III is the major product. Water (40 ml) isadded and heating is continued at an elevated temperature of about78-100° C. until the Z-isomers of anhydroluteins are converted to theirall-E-isomers and the mixture arrives at equilibrium. This isomerizationcan be followed by an HPLC method that has been previously described byKhachik et al. J. Chrom. Biomed. Appl., 670:219-233, (1995). Virtuallyall alcohols or their combinations with no limitation can be employed.These include: ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol,isobutyl alcohol, tert-butyl alcohol, 1-pentanol (n-amyl alcohol),2-n-hexyl alcohol, n-octyl alcohol, ethylene glycol, propylene glycol,propylene glycol, Some of these alcohols form an azeotropic mixture withwater and as a result the boiling point of the solutions in most casesis in the range of 78-100° C. The acid can be sulfuric acid,hydrochloric acids, phosphoric acid and the like. A strong organic acidsuch as trifluoroacetic acid may also be employed. A small amount ofZ-isomers of the anhydroluteins formed during this reaction can beremoved by simply filtering the aqueous alcoholic solution of theproduct or by isolation and crystallization of anhydroluteins prior toconversion of this carotenoid to (3R,6′R)-β-cryptoxanthin and(3R)-α-cryptoxanthin. A summary of some of the acid-catalyzeddehydration reactions of (3R,3′R,6′R)-lutein to anhydrolutein III invarious alcohols is shown in Table 2. TABLE 2 The products ofacid-catalyzed dehydration of (3R,3′R,6′R)- lutein (containing 5-7%(3R,3′R)-zeaxanthin) with sulfuric acid in various alcohols according toMethod 1. Lutein (85%), g Containing 5-7% Alcohol, ml Recovered all-E-Zeaxanthin 50% H₂SO₄/H₂O Zeaxanth Anhydroluteins (Reaction Time, (v/v)Temp in (%) h)^(a) (ml)^(b) ° C. (%) I II III 1.00 (4 h) Ethanol, 30 ml78 14 19 23 44 0.8 ml 1.00 (0.5 h) 1-Propanol, 30 ml 87 9 25 21 45 0.2ml 1.00 (2 h) 2-Propanol, 30 ml 80 12 16 23 49 0.8 ml^(a)Indicates the time needed for conversion of lutein toE/Z-anhydroluteins; an additional 4-5 h of heating under reflux isneeded to convert the E/Z-anhydroluteins to all-E-anhydroluteins.^(b)In all cases, 50% H₂SO₄/H₂O (v/v) was first added at 78-87° C. andwater (40 ml) was added after substantial amounts of E/Z-anhydroluteinIII was formed.

The work-up of the acid-catalyzed dehydration of (3R,3′R,6′R)-lutein toanhydroluteins is as follows. The reaction mixture is cooled to ambienttemperature or lower, preferably in the range of about 5-10° C., and thesolids are collected by filtration. The solids are washed with a smallamount of an alcohol (e.g. ethanol) and directly used for thepreparation of (3R,6′R)-β-cryptoxanthin and (3R)-α-cryptoxanthin in thenext step.

A more elaborate work-up consists of isolation and purification ofanhydroluteins prior to their transformation to (3R,6′R)-β-cryptoxanthinand (3R)-α-cryptoxanthin. According to this method, the crude mixture ofanhydroluteins is neutralized with an aqueous mineral base such aspotassium or sodium hydroxide and the product is dissolved in an organicsolvent immiscible with water (ethyl acetate, tert-butyl methyl ether,dichloromethane, 1,2-dichloroethane). After removing the base by washingthe organic layer with water, the solvents are evaporated and theanhydroluteins are crystallized from an alcohol.

Therefore the invention relates to a method of reacting(3R,3′R,6′R)-lutein with a catalytic amount of an acid to obtain amixture of anhydroluteins, comprising reacting (3R,3′R,6′R)-lutein withan alcohol that is also used as the solvent in the presence of catalyticamount of an acid above ambient temperature, preferably between 78-87°C., until the product is enriched in E/Z-anhydrolutein III, adding waterand further heating at this temperature until Z-isomers ofanhydroluteins are mostly converted to their all-E-counterparts. In apreferred embodiment, the reaction comprises:

-   -   a) suspending commercially available (3R,3′R,6′R)-lutein        containing 5-7% (3R,3′R)-zeaxanthin in an appropriate volume        (about 3 ml solvent/100 mg lutein) of an alcohol and adding        catalytic amount of an aqueous acid (e.g. 0.2-0.8 ml of 50%        H₂SO₄/water (v/v) per g of lutein) to obtain a mixture;    -   b) stirring the mixture at 78-87° C. for about 0.5-4 hours to        obtain a mixture of anhydroluteins rich in anhydrolutein III;    -   c) adding water (e.g. about 40 ml/g of lutein) and more aqueous        acid (e.g. about 0.4-0.8 ml of 50% H₂SO₄/water (v/v) per g of        lutein) if needed and heating the mixture between 78-87° C. to        obtain a mixture of all-E-anhydroluteins in which        all-E-anhydrolutein III is the major product;    -   d) cooling down the product to ambient temperature or below,        preferably at about 5-10° C. to obtain a crystalline mixture of        anhydroluteins;    -   e) filtering and washing the crystals with an alcohol or        acetone; collecting the crystals and transforming the crude        mixture of anhydroluteins directly to (3R)-β-cryptoxanthin and        (3R,6′R)-α-cryptoxanthin in the next step without further        purification.

Alternatively, the crude reaction mixture is worked up and the productis crystallized to give a mixture of anhydroluteins. In a preferredembodiment, the work-up and crystallization of the product comprises:

-   -   adding a base to neutralize the acid and dissolving the product        in an organic solvent immiscible with water (e.g. ethyl acetate,        tert-butyl ethyl ether, dichloromethane, 1,2-dichloroethane);    -   washing the organic layer with water to remove the base and        evaporating the solvent to obtain a concentrated residue        containing anhydroluteins;    -   crystallizing anhydroluteins from an alcohol; collecting and        drying the crystals, e.g. under high vacuum at about 60° C.

Acid-Catalyzed Dehydration of (3R,3′R,6′R)-Lutein to Anhydroluteins atElevated Temperatures by Method 2

In a typical experiment, a suspension of 85% commercially available(3R,3′R,6′R)-lutein (1.0 g of 85% pure, 0.85 g, 1.49 mmol) in 30 ml ofan alcohol such as 1-propanol is treated with 0.2 ml of 50% sulfuricacid/water (v/v) and the mixture is heated to an elevated temperature inthe range of about 45-50° C. for 1-2 h. Small amounts of a concentratedacid is added at first since the presence of excess water at thebeginning of the reaction prevents the formation of lutein 3′-alkylether. Depending on the nature of the alcohol, (3R,3′R,6′R)-lutein isconverted to its 3′-alkyl ether within 1-2 h. Water (40 ml) followed by0.4-0.8 ml of 50% sulfuric acid/water (v/v) is added and the temperatureof the mixture is increased to 78-87° C. The heating is continued untilmost of the anhydrolutein I and II are converted to anhydrolutein IIIand the mixture arrives at an equilibrium. When 1-propanol is used asthe reacting alcohol and solvent, the final product containsapproximately 84% anhydrolutein III, 10% anhydrolutein I, and 6%anhydrolutein II. Depending on the nature of the alcohol, temperature,and the concentration of the acid, the duration of the reactions mayvary between 7-20 hours. The alcohols described in method 1 can all beused. Because of the high solubility of anhydroluteins in certainalcohols at elevated temperature, the isomerization of anhydroluteins Iand II to anhydrolutein III in some cases is accompanied byE/Z(trans/cis)-isomerization. A summary of some of the acid-catalyzeddehydration reactions of (3R,3′R,6′R)-lutein to anhydrolutein III invarious alcohols by Method 2 is shown in Table 3. TABLE 3 The productsof acid-catalyzed dehydration of (3R,3′R,6′R)- lutein (containing 5-7%(3R,3′R)-zeaxanthin) with sulfuric acid in various alcohols at elevatedtemperatures according to Method 2. Lutein (85%), g Alcohol, ml all-E-Containing 5-7% H₂O, ml Recovered Anhydroluteins Zeaxanthin 50%H₂SO₄/H₂O (v/v) Reflux Zeaxanthin (%) (Reflux Time, h) (ml)^(a) Temp °C. (%) I II III 1.00 (20 h) 1-Propanol, 30 ml 87 2 10  6 82 H₂O, 40 ml0.6 ml 1.00 (7 h) 1-Propanol, 30 ml 87 7 16 11 66 H₂O, 40 ml 0.8 ml 1.00(11 h) 1-Propanol, 30 ml 87 4 35 13 48 H₂O, 40 ml 1.0 ml 1.00 (4 h)2-Propanol, 30 ml 80 9 57  9 25 H₂O, 40 ml 1.0 ml 1.00 (6 h) Ethanol, 30ml 78 7 57  8 28 H₂O, 40 ml 0.8 ml 1.00 (2 h) 1-Butanol, 30 ml 93 947^(b) 12^(b) 32^(b) H₂O, 40 ml 18% (E) 0.4 ml 14% (Z) 1.00 (2 h)2-Butanol, 30 ml 87 8 56^(b) 11^(b) 25^(b) H₂O, 40 ml 13% (E) 1.0 ml 12%(Z) 1.00 (5 h) tert-Butyl alcohol, 30 ml 83 7 48 10 35 H₂O, 40 ml 0.8 ml^(a)Indicates the total amount of acid used; in all cases, 0.2 ml of 50%H₂SO₄/H₂O (v/v) was first added at 50° C. and the remainder of the acidand water (40 ml) were added after lutein 3′-alkyl ethers were formed.^(b)Due to solubility of anhydroluteins in 1-butanol, 2-butanol, andtert-butyl alcohol at the reflux temperature, considerable amount ofZ-isomer of anhydroluteins were formed.

The work-up of the acid-catalyzed dehydration of (3R,3′R,6′R)-lutein toanhydroluteins by this method is identical to that described in Method1.

Conversion of Anhydroluteins to (3R)-β-Cryptoxanthin and(3R,6′R)-α-Cryptoxanthin by Ionic Hydrogenation with Borane-Anine/TFA

The crude or purified mixture of anhydroluteins that has a highconcentration of anhydrolutein III (prepared by methods 1 or 2) reactswith borane-amine complexes such as borane-trimethylamine (Me₃N.BH₃) orborane-dimethylamine (Me₂NH.BH₃), or borane-tert-butylamine(Me₃CNH₂.BH₃) complexes in the presence of an acid, preferablytrifluoroacetic acid (TFA), at ambient temperature in a chlorinatedsolvent (e.g. dichloromethane, 1,2-dichloroethane) to give a mixture of(3R)-β-cryptoxanthin as the major product and (3R,6′R)-α-cryptoxanthinas the minor product in excellent yields within 1-3 hours. Other hydrideion donors that can be used in the present invention includetrialkylsilanes including trimethylsilane and triethylsilane. The 5-7%of (3R,3′R)-zeaxanthin that is present in the starting material does notreact with borane-amine complex/TFA and can be recovered in the product.

In a typical experiment, a solution of anhydroluteins (1.1 mmol) indichloromethane (20 ml) is first treated with 1.37 mmol of borane-aminecomplex and this is followed by the addition of TFA (3.89 mmol). Themixture is stirred at ambient temperature for 1-3 hours. This results ina mixture of E/Z(trans/cis)-isomers of (3R)-β-cryptoxanthin and(3R,6′R)-α-cryptoxanthin. The solvent is displaced with an alcohol andwater and the mixture is heated at about 78-87° C. to convert theZ-isomers of these carotenoids to their all-E-isomer. The work-upconsists of neutralizing the acid with a base and extracting the productwith an organic solvent immiscible with water (e.g. ethyl acetate,tert-butyl ethyl ether, dichloromethane, 1,2-dichloroethane) andcrystallizing the product from an alcohol to obtainall-E-(3R)-β-cryptoxanthin (major product) andall-E-(3R,6′R)-α-cryptoxanthin (minor product).

Therefore the invention relates to a method of converting crude or apurified mixture of anhydroluteins rich in anhydrolutein III to(3R)-β-cryptoxanthin (major product) and (3R,6′R)-α-cryptoxanthin (minorproduct), comprising reacting anhydroluteins, Me₃N.BH₃ or Me₂NH.BH₃ orMe₃CNH₂.BH₃ or other borane-amine complexes, or hydride donors, and anacid in a chlorinated solvent, preferably dichloromethane, at ambienttemperature. In a preferred embodiment, the reaction comprises:

-   -   dissolving anhydroluteins containing (3R,3′R)-zeaxanthin in an        appropriate volume (e.g. about 2 ml solvent/100 mg        anhydrolutein) of dichloromethane and adding about 1.3 mol        equivalent of borane-amine complex then adding about 3.5-4 mol        equivalent of TFA to obtain a mixture;    -   stirring the mixture at ambient temperature for about 1-3 hours;    -   displacing the chlorinated solvent with an alcohol and water        heating the mixture at 78-87° C. to convert the Z-isomers of        cryptoxanthins to their all-E-counterparts;    -   adding an aqueous solution of a base (e.g. sodium bicarbonate)        to neutralize the acid, extracting with an organic solvent, and        crystallizing the residue from alcohol;    -   collecting the crystals, e.g. by filtration or on a centrifuge,        and washing the crystals with an alcohol or acetone;    -   drying the crystals, e.g. under high vacuum at about 60° C., to        obtain a mixture of recovered (3R,3′R)-zeaxanthin,        all-E-(3R)-β-cryptoxanthin (major product) and        all-E-(3R,6′R)-α-cryptoxanthin (minor product).

Extractions are performed with any organic solvent. Preferred organicsolvents include ethyl acetate, an ether, or a chlorinated solvent.

Various alcohols can be used in the present invention, including but notlimited to C₁₋₁₀ alcohols such as methanol, ethanol, 1-propanol,2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol,1-pentanol, 2-pentanol, n-hexyl alcohol, n-octyl alcohol, ethyleneglycol, and propylene glycol. Various aqueous mineral acids can be usedin the present invention, including but not limited to aqeuous sulfuric,hydrochloric, hydrobromic, hydrofluoric, nitric, and phosphoric acid.Various strong organic acids can be used in the present invention,including but not limited to trifluoroacetic, trichloroacetic, andchloroacetic acid.

“Elevated temperature” as used here is meant to be any temperature inthe range of about 45-100° C.

“Substantially no isomerization” is used herein to mean that at leastgreater than about 95% of a product or starting material is present inthe all-trans or all-E confirmation.

“Substantially no Z-isomers” is used herein to mean that at leastgreater than about 95% of a product or starting material is present inthe all-trans or all-E confirmation.

As used herein, the term “about” means that number referred to as having“about” comprises the recited number plus or minus up to 10% of thatnumber. For example, “about 5 hours” includes 4.5 to 5.5 hours. “About0° C.” includes −10° C., 0° C. and +10° C.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLE 1 Conversion of (3R,3′R,6′R)-Lutein (85% pure) to AnhydroluteinsI, II, and III with Sulfuric Acid in Ethanol Using Method 1

A suspension of (3R,3′R,6′R)-lutein (1.0 g of 85% pure, 1.49 mmol) in 30ml of ethanol was treated with 50% (v/v) sulfuric acid/water (0.80 ml)and the mixture was heated to reflux (78-80° C.). Shortly after, a darkred solution was obtained. The course of the reaction was followed byHPLC. After 4 h, water (40 ml) was added and reflux continued at 78-80°C. for 4 h until most of the Z-anhydroluteins were converted toall-E-anhydroluteins. The mixture was allowed to cool down to roomtemperature and the product was collected by filtration. The redcrystals were washed with 15 ml of ethanol, and dried under high vacuumat 60° C. to give 0.85 g of anhydroluteins (85% pure, 0.72 g, 1.3 mmol;87%) which was shown by HPLC to consist of anhydrolutein III (44%),anhydrolutein II (23%), (3R,6′R)-anhydrolutein I (19%), and unreacted(3R,3′R)-zeaxanthin (14%). This mixture was used in subsequent reactionswith Me₃N.BH₃/TFA without further purification.

EXAMPLE 2 Conversion of Anhydroluteins from Method 1 to(3R)-β-Cryptoxanthin and (3R,6′R)-α-Cryptoxanthin withMe₃N.BH₃/Trifluoroacetic Acid (TFA) in Dichloromethane

A solution of anhydroluteins prepared in Example 1 (0.85 g of 85% pure,0.72 g, 1.3 mmol) in dichloromethane (20 ml) was first treated withborane-trimethylamine complex (0.126 g, 1.73 mmol) followed bytrifluoroacetic acid (0.40 ml, 0.592 g, 5.19 mmol). The mixture wasstirred at ambient temperature and the course of the reaction wasfollowed by HPLC. After 3 h, the product was treated with 20 ml of 2%sodium bicarbonate (20 ml). The organic layer was removed, washed withwater (20 ml), and dried over sodium sulfate. The dichloromethane wasdisplaced with ethanol (30 ml) and the mixture was heated under refluxfor 2 h until most of Z(cis)-cryptoxanthins were shown by HPLC to haveconverted to their all-E(trans)-isomers and the mixture arrived at anequilibrium. The solution was allowed to cool down to room temperatureand the crystals were removed by filtration and washed with 10 ml ofcold ethanol. After drying under high vacuum at 60° C., the dark orangeproduct was shown by BPLC to contain 0.67 g (80% pure) of totalcarotenoids consisting of a mixture of (3R)-β-cryptoxanthin (55%),(3R,6′R)-α-cryptoxanthin (30%), anhydrolutein II (8%), and(3R,3′R)-zeaxanthin (7%).

EXAMPLE 3 Conversion of (3R,3′R,6′R)-Lutein (85% pure) to AnhydroluteinsI, II, and III with Sulfuiric Acid in 1-Propanol Using Method 2

A suspension of (3R,3′R,6′R)-lutein (1.0 g of 85% pure, 1.49 mmol)) in30 ml of 1-propanol was treated with 50% (v/v) sulfuric acid/water (0.20ml) and the mixture was heated to 50° C. for 1 h until a dark redsolution was obtained and all the starting material was completelyconverted to lutein 3′-propyl ether. At this time only the unreacted(3R,3′R)-zeaxanthin was shown to be present by HPLC. Water (40 ml) wasadded followed by 50% (v/v) sulfuric acid/water (0.40 ml) and thetemperature of the solution was increased to 87° C. After 20 h, themixture was allowed to cool down to room temperature and the product wastreated with 3 ml of a 9M solution of potassium hydroxide (prepared from50.5 g of KOH in 100 ml of water) and ethyl acetate (40 ml). The organiclayer was removed, washed twice with 2×40 ml of water, and dried oversodium sulfate. Nearly all of the ethyl acetate was evaporated underreduced pressure below 40° C. and the residue was treated with ethanol(30 ml) and cooled to 5-10° C. until anhydroluteins crystallized. Thecrystals were removed by filtration, washed with 15 ml of ethanol, anddried under high vacuum at 60° C. to give 0.85 g (85% pure, 0.72 g, 1.3mmol; 87%) of a dark red product which was shown by HPLC to contain amixture of anhydroluteins and recovered (3R,3′R)-zeaxanthin. Therelative distribution of carotenoids in this product was: anhydroluteinIII (82%), (3R,6′R)-anhydrolutein I (10%), anhydrolutein 11 (6%), andunreacted (3R,3′R)-zeaxanthin (2%). Substantially no Z(cis)-isomers ofanhydroluteins were detected in the final product. This mixture was usedin subsequent reactions with Me₃N.BH₃/TFA without further purification.

EXAMPLE 4 Conversion of Anhydroluteins from Method 2 to(3R)-β-Cryptoxanthin and (3R,6′R)-α-Cryptoxanthin withMe₃N.BH₃/Trifluoroacetic Acid (TFA) in Dichloromethane

A solution of anhydroluteins prepared in Example 3 (0.85 g of 85% pure,0.72 g, 1.3 mmol) in dichloromethane (20 ml) was first treated withborane-trimethylamine complex (0.114 g, 1.56 mmol) followed bytrifluoroacetic acid (0.36 ml, 0.533 g, 4.67 mmol). The mixture wasstirred at ambient temperature and the course of the reaction wasfollowed by HPLC. After 3 h, the product was treated with 20 ml of 2%sodium bicarbonate (20 ml). The organic layer was removed, washed withwater (20 ml), and dried over sodium sulfate. The dichloromethane wasdisplaced with ethanol (30 ml) and the mixture was heated under refluxfor 2 h until most of Z(cis)-cryptoxanthins were shown by HPLC to haveconverted to their all-E(trans)-isomers and the mixture arrived at anequilibrium. The solution was allowed to cool down to room temperatureand the crystals were removed by filtration and washed with 10 ml ofcold ethanol. After drying under high vacuum at 60° C., the dark orangeproduct was shown by HPLC to contain 0.60 g (80% pure) of totalcarotenoids consisting of a mixture of (3R)-β-cryptoxanthin (61%),(3R,6′R)-α-cryptoxanthin (18%), aiihydrolutein II (10%), anhydroluteinIII (7%), and (3R,3′R)-zeaxanthin (4%). II (10%).

EXAMPLE 5 Conversion of (3R,3′R,6′R)-Lutein (85% pure) to AnhydroluteinsI, II, and III with Sulfuiric Acid in 2-Propanol Using Method 1

A suspension of (3R,3′R,6′R)-lutein (1.0 g of 85% pure≈0.85 g, 1.49mmol)) in 30 ml of 2-propanol was treated with 50% (v/v) sulfuricacid/water (0.80 ml) and the mixture was heated to reflux (80-82° C.).After 2 h, water (40 ml) was added and reflux continued at 80-82° C. for4 h until most of the Z-anhydroluteins were converted toall-E-anhydroluteins. The mixture was allowed to cool down to roomtemperature and the product was treated with 6 ml of a 9M solution ofpotassium hydroxide (prepared from 50.5 g of KOH in 100 ml of water) andethyl acetate (40 ml). The organic layer was washed with water (2×30 ml)and dried over sodium sulfate. Most of the solvent was evaporated andthe residue in about 10 ml of solvent was treated with ethanol (30 ml)and allowed to stand at 5-10° C. until anhydroluteins crystallized. Thered crystals were collected by filtration, washed with 15 ml of ethanol,and dried under high vacuum at 60° C. to give 0.80 g of anhydroluteins(85% pure, 0.68 g, 1.24 mmol; 83%) which was shown by HPLC to consist ofanhydrolutein III (49%), anhydrolutein II (23%), (3R,6′R)-anhydroluteinI (16%), and unreacted (3R,3′R)-zeaxanthin (12%).

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

1. A process for converting (a) (3R,3′R,6′R)-lutein (85% totalcarotenoids) or (b) purified lutein (97% total carotenoids) containing5-7% (3R,3′R)-zeaxanthin or mixtures of (a) and (b) to a mixture ofanhydroluteins I, II, and III at elevated temperature, comprisingreacting (3R,3′R,6′R)-lutein in an alcohol or mixture of alcohols with acatalytic amount of an aqueous mineral acid or a strong organic acid atan elevated temperature to give a mixture of anhydroluteins comprisinganhydrolutein III as the major product, anhydroluteins I and II as theminor products, and the recovered (3R,3′R)-zeaxanthin.
 2. The process ofclaim 1, wherein said mixture of anhydroluteins comprises at least 80%anhydrolutein III.
 3. The process of claim 2, wherein said anhydroluteinIII comprises substantially no Z-anhydrolutein III.
 4. The process ofclaim 1, wherein said alcohol is selected from the group consisting ofethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol,tert-butyl alcohol, 1-pentanol (n-amyl alcohol), 2-pentanol, n-hexylalcohol, n-octyl alcohol, ethylene glycol, and propylene glycol.
 5. Theprocess of claim 1, wherein said aqueous mineral acid is aqueoussulfuric, hydrochloric or phosphoric acid, and said strong organic acidis trifluoroacetic or trichloroacetic acid.
 6. The process of claim 1,wherein said elevated temperature is 78-100° C.
 7. The process of claim1, further comprising adding water to convert Z(cis)-isomers ofanhydroluteins at the elevated temperature to all-E-anhydrolutein richin all-E-anhydrolutein III.
 8. The process of claim 1, furthercomprising isomerizing anhydroluteins I and II to anhydrolutein III atelevated temperatures in the presence of water and acid.
 9. The methodof claim 1, further comprising cooling the mixture of anhydroluteins toambient temperature or between 5-10° C. to precipitate theall-E-anhydroluteins as a crystalline product.
 10. The method of claim9, further comprising filtering and washing the crystalline product withan alcohol or acetone and drying at about 60° C. under high vacuum. 11.The method of claim 9, further comprising reacting all-E-anhydroluteinswith a chlorinated solvent, hydride donor and a strong organic acid togive a mixture of all-E-(3R)-β-cryptoxanthin andall-E-(3R,6′R)-α-cryptoxanthin.
 12. The method of claim 1, wherein themixture of anhydroluteins is neutralized with an aqueous or an organicbase, extracted with an organic solvent, and crystallized from analcohol.
 13. The method of claim 12, wherein the organic solvent isethyl acetate, an ether, or a chlorinated solvent.
 14. A process forconverting (a) (3R,3′R,6′R)-lutein (85% total carotenoids) or (b)purified lutein (97% total carotenoids) containing 5-7%(3R,3′R)-zeaxanthin or mixtures of (a) and (b) to lutein 3′-alkyl ether,comprising reacting (3R,3′R,6′R)-lutein with an alcohol in the presenceof a catalytic amount of an aqueous mineral acid or a strong organicacid at an elevated temperature to give all-E(trans)-lutein 3′-alkylether and the recovered (3R,3′R)-zeaxanithin.
 15. The process of claim14, wherein said alcohol is ethanol, 1-propanol, 2-propanol, 1-butanol,2-butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol (n-amylalcohol), 2-pentanol, n-hexyl alcohol, n-octyl alcohol, ethylene glycol,or propylene glycol.
 16. The process of claim 14, wherein said aqueousmineral acid is aqueous sulfuric, hydrochloric or phosphoric acid, andsaid strong organic acid is trifluoroacetic or trichloroacetic acid. 17.The process of claim 14, wherein said elevated temperature is 45-50° C.18. The method of claim 14, further comprising transforming the lutein3′-alkyl ether ill situ into a mixture of anhydroluteins, comprisingreacting lutein 3′-alkyl ether with catalytic amount of an aqueousmineral acid or a strong organic acid at an elevated temperature inwater to give anhydrolutein III as the major product and anhydroluteinsI and II as minor products.
 19. The method of claim 18, wherein theaqueous mineral acid is aqueous sulfuric, hydrochloric or phosphoricacid, and the strong organic acid is trifluoroacetic acid.
 20. Theprocess of claim 18, wherein the elevated temperature is 78° C. orhigher.
 21. The process of claim 18, further comprising adding waterthereby preventing E/Z(trais/cis)-isomerization of anhydroluteins at anelevated temperature of 78° C. or higher.
 22. The process of claim 18,further comprising isomerizing anhydroluteins I and II to anhydroluteinIII at elevated temperatures in the presence of water in 7-20 h.
 23. Themethod of claim 18, further comprising cooling said mixture ofanhydroluteins to ambient temperature or between 5-10° C. to precipitatethe all-E-anhydroluteins as a crystalline product.
 24. The method ofclaim 18, further comprising filtering said crystalline product, washingsaid product with an alcohol or acetone, and drying said product at 60°C. under high vacuum.
 25. The method of claim 24, further comprisingreacting said product with a chlorinated solvent, hydride donor and astrong organic acid to give a mixture of all-E-(3R)-β-cryptoxanthin andall-E-(3R,6′R)-α-cryptoxanthin.
 26. The method of claim 14, wherein saidall-E(tra1is)-lutein 3′-alkyl ether is neutralized with an aqueous or anorganic base, extracted with an organic solvent, and crystallized froman alcohol.
 27. The method of claim 26, wherein the organic solvent isethyl acetate, an ether or a chlorinated solvent.
 28. A process forconverting a mixture of all-E-anhydroluteins rich in all-E-anhydroluteinIII, to a mixture of all-E-(3R)-β-cryptoxanthin andall-E-(3R,6′R)-α-cryptoxanthin, comprising reacting anhydroluteinscontaining 3-8% (3R,3′R)-zeaxanthin in a chlorinated solvent with about1.3 equivalent of a hydride donor and about 3.5-4 equivalent of a strongorganic acid at ambient temperature for about 1-5 hours to give amixture of E/Z-(3R)-β-cryptoxanthin, E/Z-(3R,6′R)-α-cryptoxanthin,unreacted E/Z-anhydroluteins, and recovered E/Z-(3R,3′R)-zeaxanthin. 29.The method of claim 28, wherein said hydride ion donor isborane-trimethylamine, borane-dimethylamine, or borane-tert-butylamine.30. The method of claim 28, wherein said hydride ion donor is atrialkylsilanes.
 31. The method of claim 30, wherein saidtrialkylsilanes is triethylsilane.
 32. The method of claim 28, whereinsaid strong organic acid is trifluoroacetic acid.
 33. The method ofclaim 28, further comprising heating said mixture ofE/Z-(3R)-β-cryptoxanthin, E/Z-(3R,6′R)-α-cryptoxanthin, unreactedE/Z-anhydroluteins, and recovered E/Z-(3R,3′R)-zeaxanthin in an alcoholat 78-87° C. to convert the Z-carotenoids to their all-E-isomers andobtain a mixture of all-E-(3R)-β-cryptoxanthin,all-E-(3R,6′R)-α-cryptoxanthin, unreacted all-E-anhydroluteins, andrecovered all-E-(3R,3′R)-zeaxanthin.
 34. The method of claim 33, whereinsaid alcohol is ethanol, 1-propanol, 2-propanol.
 35. The method of claim33, further comprising neutralizing said all-E-isomers with an aqueousor an organic base, removing the aqueous layer, and displacing thechlorinated solvent with a higher boiling alcohol by distillation underreduced pressure until carotenoids crystallize from the alcohol.
 36. Themethod of claim 35, further comprising crystallizing carotenoids,collecting said carotenoids, washing said carotenoids with acetone oralcohol and drying said carotenoids under high vacuum at 60° C.